Background passive immunotherapy is a therapeutic alternative for patients with COVID-19. Equine polyclonal antibodies (EpAbs) could represent a source of scalable neutralizing antibodies against SARS-CoV-2. Methods we conducted a double-blind, randomized, placebo-controlled trial to assess efficacy and safety of EpAbs (INM005) in hospitalized adult patients with moderate and severe COVID-19 pneumonia in 19 hospitals of Argentina. Primary endpoint was improvement in at least two categories in WHO ordinal clinical scale at day 28 or hospital discharge (ClinicalTrials.gov number NCT04494984). Findings between August 1st and October 26th, 2020, a total of 245 patients were enrolled. Enrolled patients were assigned to receive two blinded doses of INM005 ( n = 118) or placebo ( n = 123). Median age was 54 years old, 65•1% were male and 61% had moderate disease at baseline. Median time from symptoms onset to study treatment was 6 days (interquartile range 5 to 8). No statistically significant difference was noted between study groups on primary endpoint (risk difference [95% IC]: 5•28% [-3•95; 14•50]; p = 0•15). Rate of improvement in at least two categories was statistically significantly higher for INM005 at days 14 and 21 of follow-up. Time to improvement in two ordinal categories or hospital discharge was 14•2 (± 0•7) days in the INM005 group and 16•3 (± 0•7) days in the placebo group, hazard ratio 1•31 (95% CI 1•0 to 1•74). Subgroup analyses showed a beneficial effect of INM005 over severe patients and in those with negative baseline antibodies. Overall mortality was 6•9% the INM005 group and 11•4% in the placebo group (risk difference [95% IC]: 0•57 [0•24 to 1•37]). Adverse events of special interest were mild or moderate; no anaphylaxis was reported. Interpretation Albeit not having reached the primary endpoint, we found clinical improvement of hospitalized patients with SARS-CoV-2 pneumonia, particularly those with severe disease.
Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.
Trypanosoma cruzi adenylyl cyclases are encoded by a large polymorphic gene family. Although several genes have been identified in this parasite, little is known about the properties and regulation of these enzymes. Here we report the cloning and characterization of TczAC, a novel member of T. cruzi adenylyl cyclase family. The TczAC gene is expressed in all of the parasite life forms and encodes a 1,313-amino acid protein that can complement a Saccharomyces cerevisiae mutant deficient in adenylyl cyclase activity. The recombinant enzyme expressed in yeasts is constitutively active, has a low affinity for ATP (K m ؍ 406 M), and requires a divalent cation for catalysis. TczAC is inhibited by Zn 2؉ and the P-site inhibitor 2-deoxyadenosine 3-monophosphate, suggesting some level of conservation in the catalytic mechanism with mammalian adenylyl cyclases. It shows a dose-dependent stimulation by Ca 2؉ which can be reversed by high concentrations of phenothiazinic calmodulin inhibitors. However, bovine calmodulin fails to stimulate the enzyme. Using a yeast two-hybrid screen it was found that TczAC interacts through its catalytic domain with the paraflagellar rod protein, a component of the flagellar structure. Furthermore, we demonstrate that TczAC can dimerize through the same domain. These results provide novel evidence of the possible localization and regulation of this protein.
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